Difference between revisions of "Part:BBa K639001"

Revision as of 14:26, 20 September 2011

relA (ppGpp synthase)

NOTE: This BioBrick has a PstI site in bp 1363, and uses OWW prefix and suffix. ( http://openwetware.org/wiki/Synthetic_Biology:BioBricks/Part_fabrication )

relA codes for ppGpp synthase, which has been shown to be useful for regulating the rrnB P1 promoter, mimicking the stringent response when over expressed [http://www.sciencedirect.com/science/article/pii/0168165695000039 Tedin, K., A. Witte, et al. (1995)]. ppGpp should effectively down-regulate the promoter by affecting the open complex's stability and inhibiting promoter clearance.

RelA is associated with about every one in two hundred ribosomes and it becomes activated when an uncharged transfer RNA (tRNA) molecule enters the A site of the ribosome, due to the shortage of amino acid required by the tRNA.

This biobrick was made from PCR amplification using cromosomal DNA as template and primers containing [http://openwetware.org/wiki/Synthetic_Biology:BioBricks/Part_fabrication Openwetware prefix and suffix];

relA.fwd: GTTTCTTCGAATTCGCGGCCGCTTCTAGAGATGGTTGCGGTAAGAAGTGCACA

relA.rev: GTTTCTTCCTGCAGCGGCCGCTACTAGTACTAACTCCCGTGCAACCGACG

The gene sequence was confirmed by sequencing, but the suffix seems to lack a PstI site.

Our project involved using the rrnB P1 promoter as a stress-sensor, where it would stop expressing an inhibitor for a second promoter, thus giving a signal. To show ppGpp's effect directly, we planned to express relA together with an rrnB P1 promoted lacZ. We were not able to show any effect due to cloning problems of the lacZ construct.

Sequence and Features